a) Field of the Invention
The invention is directed to a device for calibrating an optical detection channel for a two-dimensional, spatially dependent radiation measurement for multi-specimen carriers, particularly microtitration plates (microplates), e.g., for detecting luminescence, fluorescence, phosphorescence or radiation of radioactively labeled assays or for measuring the transmission of such specimens. It is applied especially in optical analysis devices for searching active ingredients in pharmacology and biotechnology.
b) Description of the Related Art
In biochemistry and pharmacology, it is necessary in many cases to test as many different substances as possible in microtitration plates (microplates) within a short period of time by adding reagents or cells. This is usually carried out in the form of an assay in which it is precisely determined at what time the microplate with its specimens must be at what location in what sequence. Usually, the reactions of living cells on substances of pharmacological interest are tested. For this purpose, the cells must be kept in a nutrient medium at a specified temperature and mixed with substances, kept in the incubator again for a defined period of time, etc. But the reverse is also possible, namely, the addition of substances to the wells of the microplate which are charged with reagents or cells.
In many cases, this preparatory handling concludes with optical measurement of luminescence. For this purpose, one or more reagents are added to the cells before or at the moment of measuring the light. Liquid is added to as many (or all) of the wells of the microplate as possible and, further, the light emission is measured simultaneously starting with the addition of liquid. In this connection, there are many competing demands when high plate throughput is to be achieved with automatic HTS (High Throughput Screening) or UHTS (Ultra-High Throughput Screening).
Since the generated light signals are sometimes expected over only a few seconds, a measurement of intensity with a time resolution in the range of seconds per well is required. However, the total measurement time over an entire microplate should be short.
Due to the high cost of the complex compounds of dispensing reagents, only a few microliters (μl) of a diluted solution may be used on the specimen. This means that a highly sensitive detection system is required.
It is known from U.S. Pat. Nos. 4,772,453 and 4,366,118, for example, to measure the luminescence in microplates successively well by well by means of a photomultiplier (SEV or PMT). A number of solutions for calibration are known (e.g., DE 41 14 030 C1 and DE 42 34 075 C1) for these measurement methods of individual measurement with photomultipliers. However, these solutions are directed only to sensitivity drift over time in detectors of this type because they are concerned exclusively with individual measurements. However, the essential value of eliminating falsification of measured values in order to be able to successfully carry out the intended quantitative substance analysis is clear nonetheless.
Advanced solutions use a highly sensitive CCD camera system for two-dimensional, spatially dependent acquisition of luminescence from microplates. An apparatus of this kind which reads out the luminescence from a microplate by means of a cooled CCD camera is disclosed, for example, in WO 01/07896, in which a Fresnel lens is used as a component of the imaging optics.
A CCD camera is also used in combination with fast optics and preferably with an additional light intensifier in Patent Application DE 102 36 029.4, which was not previously published. However, in spite of the time saved by simultaneous specimen measurement, these luminescence systems which are substantially better suited to the requirements of automatic HTS or UHTS must make do with the disadvantage that the sensitivity of the CCD camera is not homogeneous over the entire surface; in addition, this effect is combined with edge shadows or other imaging errors in the imaging optics used. Because of the low light yield of luminescence coupled with the demanding requirements for quantitative measurement resolution in an assay analysis, these spatially dependent falsifications of measurement values can not be tolerated.